Table 3.
In silico data obtained for the variants of unknown significance (VUS) identified in our study of familial CRC individuals
| Selected variants (VAR) | Reference splice site-dedicated analyses | Cryptic splice site-dedicated analyses | ESR-dedicated analyses | Protein-dedicated analyses | ||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Patient_ID | Genomic position (GRCh37) | Gene | Exon | Nucleotide change (cNomen) | Predicted protein change (pNomen) | Nearest reference | MES scores | SSFL scores | Potential local splice effect | Local MES scores | ∆tESRseq | ∆Hzei | ΔΨ | |||||||||||
| splice site | WT | Var | VAR vs WT | WT | Var | VAR vs WT | WT | Var | ||||||||||||||||
| Distance | Type | ∆ (%) | ||||||||||||||||||||||
| (nt) | (3’ or 5’ss) | Δ (%) | AGVGD | SIFT | MAPP | PolyPhen-2 | MutationTaster | |||||||||||||||||
| 3222 & 4932 | chr19:15273335 C>T | NOTCH3 | 32 | c.5854G>A | p.V1952M | 39 | 3’ | 11.5 | 11.5 | 0 | 89.2 | 89.2 | 0 | - | - | - | -1.78501 | -11.15 | -0.89 | C15 | Deleterious | bad | probably damaging | disease causing |
| 3222 | POLE | 25 | c.3046G>A | p.V1016M | -15 | 5’ | 9.1 | 9.1 | 0 | 82.5 | 82.5 | 0 | - | - | - | -0.74478 | 1 | -0.43 | C0 | Deleterious | bad | benign | disease causing | |
| 3308 | chr8:90965597 A>T | NBN | 11 | c.1720T>A | p.L574I | -126 | 5’ | 8.0 | 8.0 | 0 | 82.4 | 82.4 | 0 | - | - | - | 1.97427 | 33.38 | 0.18 | C0 | Tolerated | good | benign | polymorphism |
| chr12:133219838 C>T | POLE | 35 | c.4523G>A | p.R1508H | -29 | 5’ | 7.9 | 7.9 | 0 | 73.8 | 73.8 | 0 | - | - | - | -0.600279 | -3.17 | -0.08 | C0 | Tolerated | good | benign | disease causing | |
| 4932 | chr19:1221318 C>A | STK11 | 6 | c.841C>A | p.P281T | -22 | 5’ | 6.0 | 6.0 | 0 | 79.9 | 79.9 | 0 | - | - | - | -0.17437 | 7.7 | -0.72 | C0 | Tolerated | good | benign | disease causing |
| 5597 | chr5:56177843 C>G | MAP3K1 | 14 | c.2816C>G | p.S939C | 447 | 3’ | 12.0 | 12.0 | 0 | 100.0 | 100.0 | 0 | - | - | - | -0.486881 | -16.1 | 0 | C0 | Deleterious | good | benign | polymorphism |
| 9876 | chr17: 40729320 C>T | PSMC3IP | 3 | c.136G>A | p.V46M | 1 | 3’ | 12.5 | 11.7 | -6 | 87.5 | 83.6 | -4 | - | - | - | 2.0413 | 74.24 | -0.3 | C0 | Deleterious | - | possibly damaging | disease causing |
| 9876 & 8913 | chr14:69061228 G>A | RAD51B | 11* | c.1063G>A | p.A355T | 27 | 3’ | 11.8 | 11.8 | 0 | 80.2 | 80.2 | 0 | - | - | - | -1.24035 | -50.64 | - | C0 | Deleterious | - | benign | polymorphism |
| 12954 | chr1: 45797950_C>T | MUTYH | 10 | c.812G>A | p.R271Q | 33 | 3’ | 9.5 | 9.5 | 0 | 86.8 | 86.8 | 0 | - | - | - | -2.31042 | 0.88 | 0.09 | C35 | Deleterious | bad | possibly damaging | disease causing |
| chr17:56787304 G>A | RAD51C | 5 | c.790G>A | p.G264S | -48 | 5’ | 8.6 | 8.6 | 0 | 75.4 | 75.4 | 0 | New Acceptor Site? | 0 | 2.5 | -1.71397 | -59.31 | -0.21 | C0 | Tolerated | good | benign | disease causing | |
| 13072 | chr17:41234463 G>A | BRCA1 | 12 | c.4315C>T | p.L1439F | -43 | 5’ | 6.6 | 6.6 | 0 | 85.2 | 85.2 | 0 | - | - | - | -0.261141 | 8.83 | -2.67 | C0 | Tolerated | good | benign | polymorphism |
| 18843 | chr2:48027317 G>A | MSH6 | 4** | c.2195G>A | p.R732Q | -978 | 5’ | 8.9 | 8.9 | 0 | 81.6 | 81.6 | 0 | - | - | - | -0.805417 | -13.15 | 0 | C0 | Tolerated | good | benign | disease causing |
| 19673 | chr17:63554395 T>C | AXIN2 | 2 | c.344A>G | p.N115S | 460 | 3’ | 11.1 | 11.1 | 0 | 93.3 | 93.3 | 0 | New Acceptor Site? | 11.1 | 7.2 | -0.162849 | -2.19 | -0.05 | C0 | Tolerated | good | benign | disease causing |
| 21368 | chr5:56155672 A>G | MAP3K1 | 3 | c.764A>G | p.N255S | -71 | 5’ | 7.5 | 7.5 | 0 | 78.5 | 78.5 | 0 | New Acceptor Site? | 4.7 | 8.8 | -1.18661 | 6.7 | -0.04 | C0 | Tolerated | good | benign | polymorphism |
| 24447 | chr22:29130636 A>G | CHEK2 | 2 | c.74T>C | p.V25A | 80 | 3’ | 1.7 | 1.7 | 0 | 85.5 | 85.5 | 0 | - | - | -0.166788 | 36.67 | -0.07 | C0 | Tolerated | good | benign | polymorphism | |
| 11705 | chr11:108160467 G>A | ATM | 29 | c.4375G>A | p.G1459R | -62 | 5’ | 8.9 | 8.9 | 0 | 87.5 | 87.5 | 0 | - | - | 0.552026 | -27.26 | -0.09 | C15 | Deleterious | bad | probably damaging | disease causing | |
| chr2:47672694 C>G | MSH2 | 8 | c.1284C>G | p.H428Q | 8 | 3’ | 10.1 | 10.1 | 0 | 87.3 | 87.3 | 0 | - | - | 0.703136 | 22.05 | 0.05 | C0 | Tolerated | good | possibly damaging | disease causing | ||
| 13393 & 25167 | chr:8_90983460 G>A | NBN | 6 | c.643C>T | p.R215W | 59 | 3’ | 6.2 | 6.2 | 0 | 86.8 | 86.8 | 0 | - | - | -1.00071 | -16.87 | -0.09 | C0 | Tolerated | good | probably damaging | polymorphism | |
| 14963 | chr16:23647635 C>T | PALB2 | 4** | c.232G>A | p.V78I | 21 | 3’ | 10.0 | 10.0 | 0 | 90.3 | 90.3 | 0 | - | - | -0.783039 | -29.17 | 0.8 | C0 | Tolerated | good | benign | polymorphism | |
| 22953 | chr19:15278214 C>G | NOTCH3 | 29 | c.5208G>C | p.E1736D | 9 | 3’ | 7.8 | 7.8 | 0 | 84.2 | 84.2 | 0 | - | - | 0.0501789 | -10.44 | 0.22 | C35 | Deleterious | bad | benign | disease causing | |
| chr2:47630458 A>G | MSH2 | 1* | c.128A>G | p.Y43C | -84 | 5’ | 10.1 | 10.1 | 0 | 90.6 | 90.6 | 0 | New Donor Site? | 0 | 1.2 | 1.50425 | 4.7 | 2.11 | C55 | Deleterious | bad | probably damaging | disease causing | |
| chr14:68352648 T>G | RAD51B | 6 | c.515T>G | p.L172W | -58 | 5’ | 9.5 | 9.5 | 0 | 83.7 | 83.7 | 0 | - | - | 1.62772 | 67.77 | 1.31 | C0 | Deleterious | bad | possibly damaging | polymorphism | ||
| 24789 | chr5:112175625 C>T | APC | 15* | c.4334C>T | p.T1445I | 2376 | 3’ | 7.5 | 7.5 | 0 | 93.6 | 93.6 | 0 | - | - | -1.61023 | -90.13 | - | C0 | Tolerated | good | benign | polymorphism | |
| chr16:23646617 G>T | PALB2 | 4** | c.1250C>A | p.S417Y | -435 | 5’ | 8.9 | 8.9 | 0 | 87.5 | 87.5 | 0 | - | - | -0.551724 | -22.83 | 0.05 | C0 | Deleterious | bad | probably damaging | disease causing | ||
| 8913 | chr2:47601029 G>C | EPCAM | 3 | c.267G>C | p.Q89H | 83 | 3’ | 7.0 | 7.0 | 0 | 90.1 | 90.1 | 0 | - | - | -0.40237 | -28.93 | 0 | C0 | Tolerated | bad | possibly damaging | disease causing | |
In order to predict the biological impact of the 25 VUS, RNA splicing- and protein-dedicated bioinformatics analyses were performed as described under Materials and Methods. The stars indicate exons that could not be tested in our minigene assay, either because of their terminal position (*, 1st or last exons) or because of their large size (**). Results shown in bold were considered as predictive of a potential variant-induced negative biological effect. MES, MaxEntScan; SSFL, Splice Site Finder-Like; nt, nucleotide; 3’ or 5’ss, 3’ splice site or 5’ splice site; ESR, exonic splicing regulators; AGVGD, align-GVGD (C0, C15, C25, C35, C.45, C55, or C65 with C65 most likely to interfere with function and C0 least likely), SIFT, Sorting Intolerant From Tolerant (tolerated or deleterious), MAPP, Multivariate Analysis of Protein Polymorphism (good or bad), PolyPhen-2, Polymorphism Phenotyping v2 (benign, possibly damaging or probably damaging), MutationTaster (polymorphism or disease causing), CRC: colorectal cancer.
In order to predict the biological impact of the 25 VUS, RNA splicing- and protein-dedicated bioinformatics analyses were performed as described under Materials and Methods. The stars indicate exons that could not be tested in our minigene assay, either because of their terminal position (*, 1st or last exons) or because of their large size (**). Results shown in bold were considered as predictive of a potential variant-induced negative biological effect. MES, MaxEntScan; SSFL, Splice Site Finder-Like; nt, nucleotide; 3′ or 5’ss, 3′ splice site or 5′ splice site; ESR, exonic splicing regulators; AGVGD, align-GVGD (C0, C15, C25, C35, C.45, C55, or C65 with C65 most likely to interfere with function and C0 least likely), SIFT, Sorting Intolerant From Tolerant (tolerated or deleterious), MAPP, Multivariate Analysis of Protein Polymorphism (good or bad), PolyPhen-2, Polymorphism Phenotyping v2 (benign, possibly damaging or probably damaging), MutationTaster (polymorphism or disease causing), CRC: colorectal cancer
In order to predict their biological impact, RNA splicing- and protein-dedicated bioinformatics analyses were performed as described under Materials and Methods. Results shown in bold were considered as predictive of a potential variant-induced negative biological effect. MES, MaxEntScan; SSFL, Splice Site Finder-Like; nt, nucleotide; 3′ or 5’ss, 3′ splice site or 5′ splice site; ESR, exonic splicing regulators; AGVGD, align-GVGD (C0, C15, C25, C35, C.45, C55, or C65 with C65 most likely to interfere with function and C0 least likely), SIFT, Sorting Intolerant From Tolerant (tolerated or deleterious), MAPP, Multivariate Analysis of Protein Polymorphism (good or bad), PolyPhen-2, Polymorphism Phenotyping v2 (benign, possibly damaging or probably damaging), MutationTaster (polymorphism or disease causing)